Functional characterization of myo-inositol monophosphatase

ABSTRACT

This invention relates to the functional characterization of myo-inositol monophosphatase 2 (IMPA2), one of the enzymes acting in the phosphatidyl inositol signaling pathway. In particular, the present invention provides evidence that IMPA2 is associated with depression- and anxiety inducing conditions, in particular anxiety and affective disorders. In a first aspect the present invention provides the use of an IMPA2 enzyme in an assay to identify anti-anxiety or an anti-depression compounds. In particular to the use of an isolated polynucleotide encoding said IMPA2 protein, wherein said IMPA2 protein is preferably being selected from polynucleotides encoding the mouse, rat or human IMPA2 enzyme. It is thus an object of the present invention to provide a method for identifying anti-anxiety or anti-depression compounds wherein said compounds are capable of enhancing neuronal plasticity, said method comprising the steps of: a) providing a composition comprising an IMPA2 protein; b) contacting the IMPA2 protein with the test compound; and c) measuring the activity of the IMPA2 protein wherein a decrease in the IMPA2 activity in the presence of the test compound is an indicator of an anti-anxiety or anti-depression compound. In these assays the activity of the IMPA2 protein is assessed by measuring the hydrolysis of myo-inositol 1-phosphate to generate inositol and inorganic phosphate, in particular by measuring the accumulation of either myo-inositol monophosphate product in the form of radiolabeled inositol or inorganic phosphate (Pi) in the form of radiolabeled  32 Pi or in a colorimetric assay. The compositions comprising the IMPA2 protein could either be cellular extracts, whole cells or organisms expressing the IMPA2 proteins according to the invention.

This invention relates to the functional characterization ofmyo-inositol monophosphatase 2 (IMPA2), one of the enzymes acting in thephosphatidyl inositol signaling pathway. In particular, the presentinvention provides evidence that IMPA2 is associated with depression-and anxiety inducing conditions.

BACKGROUND OF THE INVENTION

The myo-inositol monophosphatase (IMPA) enzyme has an important role inthe phosphatidylinositol signaling system, catalyzing thedephosphorylation of various myo-inositol monophosphates to freemyo-inositol (Berridge and Irvine, 1989). Biochemical studies have shownthat lithium exerts an uncompetitive inhibition of the IMPA enzyme,probably by binding to and blocking metal-binding sites in the enzyme.The reduced activity of IMPA may lead to a depletion of intracellularfree myo-inositol, which is used in the re-synthesis of the signalprecursor inositol phospholipid (Berridge et al., 1989). Lithium has forseveral decades been used as a mood-stabilizer in the treatment ofmanic-depressive (bipolar) illness. However, the molecular mechanism ofthe mood-stabilizing effect has not been established. The inhibition bylithium on IMPA activity and its anti-bipolar effect appear within thesame range of concentrations and this biochemical effect remains anintriguing hypothesis for the moods stabilizing action of lithium.

It has been proposed that variations (e.g. loss-of-function orgain-of-function mutations) in the genes encoding myo-inositolmonophosphatases could either be implicated in the disturbed neuronalactivity of bipolar disorder or explain the observed variations in thetherapeutic efficacy of lithium (Steen et al., 1996). So far, two humangenes, IMPA1 and IMPA2, have been cloned and predicted to encode IMPAenzymes (McAllister at al. 1992; Sjøholt et al. 2000). Interestingly,the human IMPA2 gene is located on chromosome 18p11.2 a region that inseveral linkage studies has been indicated as a susceptibility locus forbipolar disorder. Further evidence for a possible association of IMPA2with bipolar was given in a study of the B lymphoblast cell lines frombipolar I affective disorder (BD-I) patients. It was found that thesecells from male BD-I patients have significantly lower IMPA2 mRNA levelsand elevated basal intracellular calcium levels compared with healthymale subjects (Yoon et al, 2001).

In a study to explore the possible role of this enzyme as a target forthe mood-stabilizing action of lithium in manic-depressive illness,IMPA2 knockout mice were phenotyped using a number of traditionalbehavioral tests.

The present results indicate that IMPA2 knockout mice are less prone toanxiety and depression-inducing conditions and point to a possible rolefor IMPA2 in affective disorders, in particular to a role in theimpaired neuroplasticity and cellular resilience found in severe moodand anxiety disorders.

The functional characterization by the present invention of IMPA2 as agene involved in neuronal plasticity provides the means to identifycompounds useful in the treatment of patients that have an impairedcapability of neuronal cells to make a long term alteration of itscircuitery and functionally in response to new inputs (learning), aswell as in the treatment of patients that have an impaired capability ofthe neuronal tissue to recover from injury by reorganizing its functionto compensate for partial destruction of tissue or loss of functioncaused be degenerative disorders.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides the use of an IMPA2enzyme in an assay to identify anti-anxiety or anti-depressioncompounds, wherein said anti-anxiety or anti-depression compounds arecapable of enhancing neuronal plasticity. Consequently, in a furtheraspect the present invention provides the use of an IMPA2 enzyme in anassay to identify compounds capable to enhance the neuronal plasticityin the CNS of a mammal.

It is thus an object of the present invention to provide a method foridentifying anti-anxiety or anti-depression compounds wherein saidanti-anxiety or anti-depression compounds are capable of enhancingneuronal plasticity, said method comprising the steps of:

a) providing a composition comprising an IMPA2 protein;

b) contacting the IMPA2 protein with the test compound; and

c) measuring the activity of the IMPA2 protein wherein a decrease in theIMPA2 activity in the presence of the test compound is an indicator ofan anti-anxiety or anti-depression compound.

In a second aspect, the present invention provides a method fordetermining whether a compound is a capable of enhancing neuronalplasticity, said method comprising the steps of;

a) providing a composition comprising an IMPA2 protein;

b) contacting the IMPA2 protein with the test compound; and

c) measuring the activity of the IMPA2 protein wherein a decrease in theIMPA2 activity in the presence of the test compound is an indicator of aneuronal plasticity enhancing compound.

In a third aspect, the invention provides the use of a compoundidentified using an assay according to the invention, in the preparationof a medicament for treating anxiety or in the preparation of amedicament for promoting neuronal plasticity, in particular in thepreparation of a medicament to enhance memory or to treat memorydysfunction, as well as to treat neuronal damage of the following kinds:stroke, multi-infarct dementia, head trauma, cerebral ischemia, braininjury, including (without limitation) injury casude by assault,accident, tumour (e.g. a brain tumour or a non-brain tumour that affectsthe brain, such as bony tumour of the skill that impinges on the brain)or surgery to remove tumours or to treat epilepsy; multiple sclerosis;and neurodegenerative diseases which affect the cortex, such as seniledementia, Alzheimer's disease, Parkinsons's disease, Huntington'schorea, cerebellar-spinal adrenoleucodystrophy, Pick's disease orWilson's disease.

In a fourth aspect the invention provides a method of treatment of acondition associated with an impaired neuronal adaptive response, suchas for example in the treatment of memory dysfunction, as well as totreat neurodegenerative diseases which affect the cortex, such as seniledementia, Alzheimer's disease, Parkinsons's disease, Huntington'schorea, cerebellar-spinal adrenoleucodystrophy, Pick's disease orWilson's disease, comprising the step of administering an effectiveamount of an IMPA2 inhibitor to a subject in need of such treatment.

It is also an object of the present invention to provide a method oftreating neurological conditions for which neuronal plasticity enhancingtreatments are envisaged, such as for example to enhance memory andlearning, as well as to treat neuronal damage of the following kinds:stroke, multi-infarct dementia, head trauma, cerebral ischemia, braininjury, including (without limitation) injury casude by assault,accident, tumour (e.g. a brain tumour or a non-brain tumour that affectsthe brain, such as bony tumour of the skill that impinges on the brain)or surgery to remove tumours or to treat epilepsy; multiple sclerosis;and neurodegenerative diseases which affect the cortex, such as seniledementia, Alzheimer's disease, Parkinsons's disease, Huntington'schorea, cerebellar-spinal adrenoleucodystrophy, Pick's disease orWilson's disease, comprising the step of administering an effectiveamount of an IMPA2 inhibitor to a subject in need of such treatment.

In a final aspect, the present invention provides the use of IMPA2 knockout animals as a model to study the effects of enhanced neuronalplasticity. In particular to study the effects of an increased adaptiveresponse to a stressor in an animal model. Such transgenic animals canbe commercially marketed to researchers, among other uses.

This and further aspects of the present invention will be discussed inmore detail hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the vector VICTR48 used to generatethe IMPA2 knockout from OST203987.

FIG. 2: Expression levels of IMPA1 and IMPA2 in different mouse tissuesamples. The expression levels in the different mouse tissues areexpressed as relative levels after normalization to mouse β-actin.

FIG. 3: Expression levels of IMPA1 and IMPA2 in different mouse tissuesamples. The expression levels in the different mouse tissues areexpressed as average cycle treshold (CT)-values.

FIG. 4: Results of the different parameters monitored in the ElevatedZero Maze test, i.e. the total distance moved, the relative duration inthe open arms and the relative distance in the open arms.

FIG. 5: Results of the different parameters monitored in the firstsession of the Porsolt forced swim test, i.e. relative immobilityduration during the first 180 sec, relative immobility during the last180 sec and the relative duration of the immobility throughout the test.

FIG. 6: Results of the second session of the Porsolt forced swim test.The same parameters were recorded.

FIG. 7: Results of the different parameters monitored in the Open Fieldtest, i.e. time spent in center, distance travelled in center, totaldistance travelled, number of moves, duration of moves, number ofrearings and duration of rearings

FIG. 8: Expression level of MIP synthase in nonstressed vs. stressedImpa2 KO mice and WT littermates. A significant effect of genotype andof stress on expression levels was found. The expression levels areexpressed as relative levels after normalization to mouse β-actin.

FIG. 9: Expression level of BDNF in nonstressed vs. stressed Impa2 KOmice and WT littermates. A significant effect of genotype and of stresson expression levels was found. The expression levels are expressed asrelative levels after normalization to mouse β-actin.

This invention will be better understood by reference to theExperimental Details that follow, but those skilled in the art willreadily appreciate that these are only illustrative of the invention asdescribed more fully in the claims that follow thereafter. Additionally,throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides the use of an IMPA2enzyme in an assay to identify anti-anxiety or anti-depressioncompounds, wherein said anti-anxiety or anti-depression compounds arecapable of enhancing neuronal plasticity. Consequently, in a furtheraspect the present invention provides the use of an IMPA2 enzyme in anassay to identify compounds capable to enhance the neuronal plasticityin the CNS of a mammal. The IMPA2 protein or functional fragment as usedherein refers to an isolated protein capable of hydrolysing myo-inositol1-phosphate to generate inositol and inorganic phosphate. It ispreferably selected from the group consisting of;

i. mouse IMPA2 (SEQ ID No:4), rat IMPA2 (SEQ ID No:6), human IMPA2 (SEQID No:2) or a functional fragment thereof, or

ii. an amino acid sequence encoding an IMPA2 protein, wherein said aminoacid sequence has at least 80% sequence identity, preferably at least90% sequence identity, more preferably at least 95% or most preferablyat least 98% sequence identity with the human IMPA2 protein (SEQ IDNo:2) over its entire length.

In particular to the use of an isolated polynucleotide encoding saidIMPA2 protein in an assay according to the invention, wherein said IMPA2protein is preferably being selected from;

i. polynucleotides encoding the mouse (EMBL:BC011093—SEQ ID No:3), rat(EMBL:AY160191—SEQ ID No:5) or human (EMBL:BC011093—SEQ ID No:1) IMPA2enzyme; or

ii. a polynucleotide sequence encodig an IMPA2 protein, wherein saidamino acid sequence has at least 80% sequence identity, preferably atleast 90% sequence identity, more preferably at least 95% or mostpreferably at least 98% sequence identity with the polynucleotideencoding for the human IMPA2 protein (SEQ ID No:1) over its entirelength.

“Neuronal plasticity”, “plasticity”, “neuroplasticity” and the like, asused herein refers to the ability of the nervous system to change and/orto develop connections between neurons so as to alter the function ofthe brain or spinal cord, often in response to sensory or behaviouralstimuli or damage. The term encompasses neurogenesis, the activation ofsynapses that were structurally present but inactive, the strengtheningand weakening of synapses, and the making and breaking of synapses.

There are many neurological conditions for which neuronal plasticityenhancing treatments are under investigation. For example, to enhancememory or to treat memory dysfunction, as well as to treat neuronaldamage of the following kinds: stroke, multi-infarct dementia, headtrauma, cerebral ischemia, brain injury, including (without limitation)injury casude by assault, accident, tumour (e.g. a brain tumour or anon-brain tumour that affects the brain, such as bony tumour of theskill that impinges on the brain) or surgery to remove tumours or totreat epilepsy; multiple sclerosis; and neurodegenerative diseases whichaffect the cortex, such as senile dementia, Alzheimer's disease,Parkinsons's disease, Huntington's chorea, cerebellar-spinaladrenoleucodystrophy, Pick's disease or Wilson's disease.

It is thus an object of the present invention to provide a method foridentifying anti-anxiety or anti-depression compounds wherein saidanti-anxiety or anti-depression compounds are capable of enhancingneuronal plasticity, said method comprising the steps of:

a) providing a composition comprising an IMPA2 protein;

b) contacting the IMPA2 protein with the test compound; and

c) measuring the activity of the IMPA2 protein wherein a decrease in theIMPA2 activity in the presence of the test compound is an indicator ofan anti-anxiety or anti-depression compound.

In a further aspect, the present invention provides a method fordetermining whether a compound is a capable of enhancing neuronalplasticity, said method comprising the steps of;

a) providing a composition comprising an IMPA2 protein;

b) contacting the IMPA2 protein with the test compound; and

c) measuring the activity of the IMPA2 protein wherein a decrease in theIMPA2 activity in the presence of the test compound is an indicator of aneuronal plasticity enhancing compound.

The compositions comprising the IMPA2 protein could either be cellularextracts, whole cells or organisms expressing the IMPA2 proteinsaccording to the invention. In a particular embodiment the compositioncomprising an IMPA2 protein consists of whole cells expressing IMPA2,more particular of CHO cells expressing IMPA2.

Typically the contacting is effected from about 1 minute to about 24hours, preferably from about 2 minutes to about 1 hour, more preferablythe contacting is effected for 1 hour.

In these assays the activity of the IMPA2 protein is assessed bymeasuring the hydrolysis of myo-inositol 1-phosphate to generateinositol and inorganic phosphate, in particular by measuring theaccumulation of either myo-inositol monophosphate product in the form ofradiolabeled inositol or inorganic phosphate (Pi) in the form ofradiolabeled ³²Pi or in a colorimetric assay. For example, a Pi-releaseassay based on colorimetric means to measure changes in Pi concentrationover time can be carried out as described by Ragan (1988) Biochem. J.249:143-148, or, by Vadnal (1995) Neuropsychopharmacol. 12:277-285.

As in Vadnal (1995) supra, the reaction mixture can consist of 0.05 mlof 120 mM Tris-HCI, pH 7.8; 0.05 ml of 18 mM or 3 mM magnesium chloride;0.05 ml of 4.2 mM D-myo-inositol 1-phosphate, 0.125 ml water alone orwith positive controls or putative modulator test compounds orcompositions. Known myo-inositol monophosphatase inhibitors(antagonists), such as lithium, carbamazepine and/or valproic acid, invarying amounts can be used as controls. A 0.025 ml solution ofmyo-inositol monophosphatase (e.g., human IMPA2) is added and thereaction mixture is incubated at 37° C. for about 15 minutes to an hour.The reaction is stopped by the addition of 0.05 ml of 20%trichloroacetic acid TCA). The suspension is centrifuged and 0.10 ml ofsupernatant is used to estimate the liberated Pi using the malachitegreen reagent method, as, for example, described by Eisenberg (1987)Methods Enzymol. 141:127-143. Protein is assayed using the method ofLowry (1951) J. Biol Chem. 193:265-275.

Assays are usually run in triplicate. Alternatively, as in Ragan (1988)supra, the reaction mixture can be in a final volume of 0.300 mlcontaining 0.1 mM substrate, 250 mM potassium chloride, 50 mM Tris HCl,pH 8.0, and 3 mM magnesium chloride for period of time from 15 minutesto one hour. Released Pi can be measured colorimetrically using themethod of Itaya (1966) Olin. Chem. Acta 14:361-366 (see also Kodama(1986) “The initial phosphate burst in ATP hydrolysis by myosin andsubfragment-1 as studied by a modified malachite green method fordetermination of inorganic phosphate,” J Biochem. (Tokyo) 99:1465-1472).The specific activity of myo-inositol monophosphatase is expressed asnanomoles of phosphate liberated per minute (mU) per milligram protein.

Kinetic activity and assessment of potential modulators of the IMPA2protein of the invention can also be accomplished in vitro and in vivoby measuring accumulation of the substrate myo-inositol monophosphate(myo-inositol I-phosphate) using, for example, assays described by Atack(1993) J. Neurochem. 60:652-658; or, Ragan (1988) supra. Radiolabeledinositol monophosphate accumulation can be measured in tissue culturecells expressing IMPA2 protein in the presence of putative myo-inositolmonophosphatase antagonists, for example, as described by Atack (1993)supra. The tissue culture cells can be genetically manipulated, asdescribed hereinafter, to express the IMPA2 protein of the invention, orfragments or variations thereof.

For example, as described above, CHO cells can be manipulated to expressvery large amounts of exogenous protein. Specifically, to assess theeffect of a putative antagonist or agonist on myo-inositolmonophosphatase in vivo, CHO cells are first pre-labeled with ³H-inositol. Prelabeling involves growing cells to confluence for twodays in medium containing radiolabeled inositol (e.g., ¹⁴ C-inositol or³ H-inositol). If using 3H-inositol, 0.5 uCi/mI 80 Ci/mmol (AmershamInternational) is used. On the day of the experiment, cells areharvested in Krebs-Henselcit buffer at 2×10⁶ cells/ml containing 0.5UCi/mI ³ H-inositol.

Aliquots of the harvested cells are incubated for one hour at 37° C. ina shaking water bath in the presence of 10 ul of various concentrationsof known enzyme inhibitors and test compounds—putative enzymemodulators. Assays are terminated by addition of 300 ul of 1.0 M TCA andcentrifuged. 500 ul of supernatant is washed with water-saturateddiethyl ether. The pH is adjusted to about 7.0 using 1 M Tris. Thesupernatants are then applied to Dowex columns. Columns are washed fourtimes with 5 ml of water to elute free ³ H-inositol; then washed threetimes with 5 ml of 25 mM ammonium formate to elutebeta-glycerophosphates. ³ H-inositol 1-monophosphate is collected bywashing the column with 10 ml of 200 mM ammonium phosphate and countedon a scintillation counter.

Alternatively, ¹⁴ C-inositol can be used, as described by Ragan (1988)supra. Inhibition of the myo-inositol monophosphatase will result inincreased levels of the substrate myo-inositol monophosphate(myo-inositol 1-phosphate), while activation of the enzyme will resultin decreased levels of substrate and increased levels of product(inositol and inorganic phosphate).

Using these assays and variations thereof, the kinetics of the IMPA2enzyme with and without test modulators (e.g., competitive ornon-competitive antagonists) can be analyzed using known methods (e.g.,Lineweaver-Burke plots, as used, for example by Lee (1996) Xenobiotica26: 8′) 1-83) 8); for discussion on enzyme kinetic analysis generallysee, for example, Suarez (1997) Proc. Nad. Acad Sci. USA 94:7065-7069;Northrop (1997) Bioorg. Med Chem. 5:641-644); Sterrer (1997) J. Recept.Signal Transduct. Res. 17:511-520).

It is thus an object of the present invention to provide the use oftissue culture cells such as for example CHO or HEK293 cells,genetically manipulated to express IMPA2, in an assay according to theinvention. Cells suitable for performing an assay according to theinvention are preferably higher eukaryotic cells derived from amulticellular organism and advantageously are mammalian cells. Cells maybe transformed by any suitable technique available in the art. A numberof techniques, such as calcium phosphate precipitation andelectroporation are described in Sambrook et al., (1989) MolecularBiology: A Laboratory Manual, Cold Spring Harbor, which is incorporatedherein by reference.

In another aspect, the invention provides the use of a compoundidentified using an assay according to the invention, in the preparationof a medicament for treating anxiety or in the preparation of amedicament for promoting neuronal plasticity, in particular in thepreparation of a medicament to enhance memory or to treat memorydysfunction, as well as to treat neuronal damage of the following kinds:stroke, multi-infarct dementia, head trauma, cerebral ischemia, braininjury, including (without limitation) injury casude by assault,accident, tumour (e.g. a brain tumour or a non-brain tumour that affectsthe brain, such as bony tumour of the skill that impinges on the brain)or surgery to remove tumours or to treat epilepsy; multiple sclerosis;and neurodegenerative diseases which affect the cortex, such as seniledementia, Alzheimer's disease, Parkinsons's disease, Huntington'schorea, cerebellar-spinal adrenoleucodystrophy, Pick's disease orWilson's disease.

Methods and pharmaceutical carriers for preparation of pharmaceuticalcompositions are well known in the art, as set out in textbooks such asRemington's Pharmaceutical Sciences, 20^(th) Edition, Williams &Wilkins, Pensylvania, USA.

The compounds and compositions of the invention may be administered byany suitable route, and the person skilled in the art will readily beable to determine the most suitable route and dose for the condition tobe treated. Dosage will be at the discretion of the attended physicianor veterinarian, and will be dependent on the state and nature of thecondition to be treated, the age and general state of health of thesubject to be treated, the route of administration, and any previoustreatment which may have been administered. The compound identified asan anti-anxiety compound or a neuroplasticity enhacing compound using anassay according to the invention may optionally be administered inconjunction with one or more other pharmaceutically active agentsuitable for the treatment of the condition, i.e. it may be giventogether, before or after one or more such agents. For example, wherethe condition involves Alzheimer's disease, the compounds may be used inconjunction with treatment with another agent such as anacetyl-cholinesterase active site inhibitor, for example phenserine,galantamine or tracine.

The carrier or diluent, and other excipients, will depend on the routeof administration, and again the person skilled in the art will readilybe able to determine the most suitable formulation for each particularcase. The compound of the invention may be administered orally,topically, or parenterally in dosage unit formulations containingconventional pharmaceutically acceptable carriers, adjuvants, andvehicles. The term parenteral as used herein includes subcutaneous,intravenous, intramuscular, intrathecal, intracranial, injection orinfusion techniques. Of particular interest is administration of thecompound to the CNS, through the blood brain barrier. The preferredroute of administration will be by direct administration to the CNS,e.g. infusion via canulla or injection. Such administration may bedirectly into the site of injury, into neighbouring tissues or into thecerebrospinal fluid.

The invention includes various pharmaceutical compositions useful forameliorating disease. The pharmaceutical compositions according to oneembodiment of the invention are prepared by bringing a compound of theinvention and optionally one or more other pharmaceutically-activeagents or combinations of the compound-of the invention and one or moreother pharmaceutically-active agents into a form suitable foradministration to a subject, using carriers, excipients and additives orauxiliaries.

Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 20th ed. Williams & Wilkins (2000) and The British NationalFormulary 43rd ed. (British Medical Association and Royal PharmaceuticalSociety of Great Britain, 2002; http://bnf.rhn.net), the contents ofwhich are hereby incorporated by reference. The pH and exactconcentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed., 1985).

The pharmaceutical compositions are preferably prepared and administeredin dosage units. Solid dosage units include tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the subject, different daily doses can be used.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

In a furter aspect the invention provides a method of treatment of acondition associated with an impaired neuronal adaptive response, suchas for example in the treatment of memory dysfunction, as well as totreat neurodegenerative diseases which affect the cortex, such as seniledementia, Alzheimer's disease, Parkinsons's disease, Huntington'schorea, cerebellar-spinal adrenoleucodystrophy, Pick's disease orWilson's disease, comprising the step of administering an effectiveamount of an IMPA2 inhibitor to a subject in need of such treatment.

It is also an object of the present invention to provide a method oftreating neurological conditions for which neuronal plasticity enhancingtreatments are envisaged, such as for example to enhance memory andlearning, as well as to treat neuronal damage of the following kinds:stroke, multi-infarct dementia, head trauma, cerebral ischemia, braininjury, including (without limitation) injury casude by assault,accident, tumour (e.g. a brain tumour or a non-brain tumour that affectsthe brain, such as bony tumour of the skill that impinges on the brain)or surgery to remove tumours or to treat epilepsy; multiple sclerosis;and neurodegenerative diseases which affect the cortex, such as seniledementia, Alzheimer's disease, Parkinsons's disease, Huntington'schorea, cerebellar-spinal adrenoleucodystrophy, Pick's disease orWilson's disease, comprising the step of administering an effectiveamount of an IMPA2 inhibitor to a subject in need of such treatment.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacological and/or physiological effect. The effect may beprophylactic in terms of completey or partially preventing a disease orsign or symptom thereof, and/or may be therapeutic in terms of a partialor complete cure of a disease. “Treating” as used herein covers anytreatment of, or prevention of disease in a vertebrate, a mammal,particularly a human, and includes: preventing the disease fromoccurring in a subject which may be predisposed to the disease, but hasnot yet been diagnosed as having it, inhibiting the disease, i.earresting its development; or relieving or ameliorating the effects ofthe disease, i.e. causing regression of the effects of the disease.

As used herein, the term “effective amount” means an amount of acompound of the present invention effective to yield a desiredtherapeutic response, for example to prevent or treat a disease which issuspectible to treatment by administration of a pharmaceuticalcomposition comprising a compound of the present invention as activeingredient. The specific “therapeutically effective amount” will be atthe discretion of the attendant physician or veterinarian and will ofcourse vary with such factors as the particular condition being treated,the physical condition and clinical history of the subject, the type ofanimal being treated, the duration of the treatment, the nature ofconcurrent therapy (if any) and the specific formulations employed.

Also described herein are non-human animals and cells which harbor atleast one integrated targeting construct that functionally disrupts anendogenous IMPA2 gene locus in said non-human animal or cell, typicallyby deleting or mutating a genetic element, e.g. exon sequence, splicingsignal, promoter enhancer, that is required for efficient functionalexpression of the IMPA2 gene product. In this embodiment, a portion ofthe targeting construct integrates into an essential structural orregulatory element of the endogenous IMPA2 gene locus, therebyfunctionally disrupting it to generate a null allele. Typically, nullalleles are produced by integrating a non-homologous sequence encoding aselectable marker (e.g. a neo gene expression cassette) into anessential structural and/or regulatory sequence of an IMPA2 gene byhomologous recombination of the targeting construct homology clamps withendogenous IMPA2 gene sequences, although other strategies may beemployed.

Most usually, a targeting construct is transferred by electroporation ormicroinjection into a totipotent embryonal stem (ES) cell line, such asthe murine AB-1 or CCE lines. The targeting construct homologouslyrecombines with endogenous sequences in or flanking an IMPA2 gene locusand functionally disrupts at least one allele of the IMPA2 gene.Typically, homologous recombination of the targeting construct withendogenous IMPA2 locus sequences results in integration of anonhomologous sequence encoding and expressing a selectable marker, suchas neo, usually in the form of a positive selection cassette. Thefunctionally disrupted allele is termed an IMPA2 null allele. ES cellshaving at least one IMPA2 null allele are selected for by propagatingthe cells in a medium that permits the preferential propagation of cellsexpressing the selectable marker. Selected ES cells are examined by PCRanalysis and/or Southern blot analysis to verify the presence of acorrectly targeted IMPA2 allele. Breeding of nonhuman animals which areheterozygous for a null allele may be performed to produce nonhumananimals homozygous for said null allele, so-called “knockout” animals(Donehower et al. (1992) Nature256: 215; Science256: 1392, incorporatedherein by reference). Alternatively, ES cells homozygous for a nullallele having an integrated selectable marker can be produced in cultureby selection in a medium containing high levels of the selection agent(e.g., G418 or hygromycin). Heterozygosity and/or homozygosity for acorrectly targeted null allele can be verified with PCR analysis and/orSouthern blot analysis of DNA isolated from an aliquot of a selected EScell clone and/or from tail biopsies.

Several gene targeting techniques have been described, including but notlimited to: co-electroporation, “hit-and-run”, single-crossoverintegration, and double-crossover recombination (Bradley et al. (1992)Bio/Technology 10: 534). The invention can be practiced usingessentially any IMPA2licable homologous gene targeting strategy known inthe art. The configuration of a targeting construct depends upon thespecific targeting technique chosen. For example, a targeting constructfor single-crossover integration or “hit-and-run” targeting need onlyhave a single homology clamp linked to the targeting region, whereas adouble-crossover replacement-type targeting construct requires twohomology clamps, one flanking each side of the replacement region.

For example and not limitation, a preferred embodiment is a targetingconstruct comprising, in order: (1) a first homology clamp having asequence substantially identical to a sequence within about 3 kilobasesupstream (i.e., in the direction opposite to the translational readingframe of the exons) of an exon of an endogenous IMPA2 gene, (2) areplacement region comprising a positive selection cassette having apgkpromoter driving transcription of a neogene, (3) a second homologyclamp having a sequence substantially identical to a sequence withinabout 3 kilobases downstream of said exon of said endogenous IMPA2 gene,and (4) a negative selection cassette, comprising a HSV tkpromoterdriving transcription of an HSV tkgene. Such a targeting construct issuitable for double- crossover replacement recombination which deletes aportion of the endogenous IMPA2 locus spanning said exon and replaces itwith the replacement region having the positive selection cassette. Ifthe deleted exon is essential for expression of a functional IMPA2 geneproduct, the resultant exon-depleted allele is functionally disruptedand is termed a null allele.

Targeting constructs of the invention comprise at least one IMPA2homology clamp linked in polynucleotide linkage (i.e., by phosphodiesterbonds) to a targeting region. A homology clamp has a sequence whichsubstantially corresponds to, or is substantially complementary to, anendogenous IMPA2 gene sequence of a nonhuman host animal, and maycomprise sequences flanking the IMPA2 gene.

Although no lower or upper size boundaries for recombinogenic homologyclamps for gene targeting have been conclusively determined in the art,the best mode for homology clamps is believed to be in the range betweenabout 50 basepairs and several tens of kilobases. Consequently,targeting constructs are generally at least about 50 to 100 nucleotideslong, preferably at least about 250 to 500 nucleotides long, morepreferably at least about 1000 to 2000 nucleotides long, or longer.Construct homology regions (homology clamps) are generally at leastabout 50 to 100 bases long, preferably at least about 100 to 500 baseslong, and more preferably at least about 750 to 2000 bases long. It isbelieved that homology regions of about 7 to 8 kilobases in length arepreferred, with one preferred embodiment having a first homology regionof about 7 kilobases flanking one side of a replacement region and asecond homology region of about 1 kilobase flanking the other side ofsaid replacement region. The length of homology (i.e., substantialidentity) for a homology region may be selected at the discretion of thepractitioner on the basis of the sequence composition and complexity ofthe endogenous IMPA2 gene target sequence(s) and guidance provided inthe art (Hasty et al. (1991) Mol. Cell. Biol. 11: 5586; Shulman et al.(1990) Mol. Cell. Biol. 10: 4466). Targeting constructs have at leastone homology region having a sequence that substantially corresponds to,or is substantially complementary to, an endogenous IMPA2 gene sequence(e.g., an exon sequence, an enhancer, a promoter, an intronic sequence,or a flanking sequence within about 3-20 kb of a IMPA2 gene). Such atargeting transgene homology region serves as a template for homologouspairing and recombination with substantially identical endogenous IMPA2gene sequence(s). In targeting constructs, such homology regionstypically flank the replacement region, which is a region of thetargeting construct that is to undergo replacement with the targetedendogenous IMPA2 gene sequence (Berinstein et al. (1992) Mol. Cell.Biol. 12: 360). Thus, a segment of the targeting construct flanked byhomology regions can replace a segment of an endogenous IMPA2 genesequence by double-crossover homologous recombination. Homology regionsand targeting regions are linked together in conventional linearpolynucleotide linkage (5′ to 3′ phosphodiester backbone). Targetingconstructs are generally double-stranded DNA molecules, most usuallylinear.

Without wishing to be bound by any particular theory of homologousrecombination or gene conversion, it is believed that in such adouble-crossover replacement recombination, a first homologousrecombination (e.g., strand exchange, strand pairing, strand scission,strand ligation) between a first targeting construct homology region anda first endogenous IMPA2 gene sequence is accompanied by a secondhomologous recombination between a second targeting construct homologyregion and a second endogenous IMPA2 gene sequence, thereby resulting inthe portion of the targeting construct that was located between the twohomology regions replacing the portion of the endogenous IMPA2 gene thatwas located between the first and second endogenous IMPA2 genesequences. For this reason, homology regions are generally used in thesame orientation (i.e., the upstream direction is the same for eachhomology region of a transgene to avoid rearrangements).Double-crossover replacement recombination thus can be used to delete aportion of an endogenous IMPA2 gene and concomitantly transfer anonhomologous portion (e.g., a neogene expression cassette) into thecorresponding chromosomal location. Double-crossover recombination canalso be used to add a nonhomologous nortion into an endogenous IMPA2gene without deleting endogenous chromosomal portions. However,double-crossover recombination can also be employed simply to delete aportion of an endogenous IMPA2 gene sequence without transferring anonhomologous portion into the endogenous IMPA2 gene (see Jasin et al.(1988) Genes Devel.2:1353). Upstream and/or downstream from thenonhomologous portion may be a gene which provides for identification ofwhether a double-crossover homologous recombination has occurred; such agene is typically the HSV tkgene which may be used for negativeselection.

Typically, targeting constructs of the invention are used forfunctionally disrupting endogenous IMPA2 genes and comprise at least twohomology regions separated by a nonhomologous sequence which contains anexpression cassette encoding a selectable marker, such as neo (Smith andBerg (1984) Cold Spring Harbor Symp. Quant. Biol. 49: 171; Sedivy andSharp (1989) Proc. Natl. Acad. Sci. (U.S.A.)86: 227; Thomas and Capecchi(1987) op.cit. ). However, some targeting transgenes of the inventionmay have the homology region(s) flanking only one side of anonhomologous sequence. Targeting transgenes of the invention may alsobe of the type referred to in the art as “hit-and-run” or “in-and-out”transgenes (Valancius and Smithies (1991) Mol. Cell. Biol.11: 1402;Donehower et al. (1992) Nature 356: 215; (1991) J. NIH Res. 3: 59; whichare incorporated herein by reference).

The positive selection expression cassette encodes a selectable markerwhich affords a means for selecting cells which have integratedtargeting transgene sequences spanning the positive selection expressioncassette. The negative selection expression cassette encodes aselectable marker which affords a means for selecting cells which do nothave an integrated copy of the negative selection expression cassette.Thus, by a combination positive-negative selection protocol, it ispossible to select cells that have undergone homologous replacementrecombination and incorporated the portion of the transgene between thehomology regions (i.e., the replacement region) into a chromosomallocation by selecting for the presence of the positive marker and forthe absence of the negative marker.

Preferred expression cassettes for inclusion in the targeting constructsof the invention encode and express a selectable drug resistance markerand/or a HSV thymidine kinase enzyme. Suitable drug resistance genesinclude, for example: gpt(xanthine-guanine phosphoribosyltransferase),which can be selected for with mycophenolic acid; neo(neomycinphosphotransferase), which can be selected for with G418 or hygromycin;and DFHR (dihydrofolate reductase), which can be selected for withmethotrexate (Mulligan and Berg (1981) Proc. Natl. Acad. Sci.(U.S.A.)78: 2072; Southern and Berg (1982) J. Mol. IMPA21. Genet. 1:327; which are incorporated herein by reference).

Selection for correctly targeted recombinants will generally employ atleast positive selection, wherein a nonhomologous expression cassetteencodes and expresses a functional protein (e. g., neoor gpt) thatconfers a selectable phenotype to targeted cells harboring theendogenously integrated expression cassette, so that, by addition of aselection agent (e.g., G418 or mycophenolic acid) such targeted cellshave a growth or survival advantage over cells which do not have anintegrated expression cassette. It is preferable that selection forcorrectly targeted homologous recombinants also employ negativeselection, so that cells bearing only nonhomologous integration of thetransgene are selected against. Typically, such negative selectionemploys an expression cassette encoding the herpes simplex virusthymidine kinase gene (HSV tk) positioned in the transgene so that itshould integrate only by nonhomologous recombination. Such positioninggenerally is accomplished by linking the HSV tkexpression cassette (orother negative selection cassette) distal to the recombinogenic homologyregions so that double-crossover replacement recombination of thehomology regions transfers the positive selection expression cassette toa chromosomal location but does not transfer the HSV tkgene (or othernegative selection cassette) to a chromosomal location. A nucleosideanalog, gancyclovir, which is preferentially toxic to cells expressingHSV tk, can be used as the negative selection agent, as it selects forcells which do not have an integrated HSV tkexpression cassette. FIAUmay also be used as a selective agent to select for cells lacking HSVtk.

In order to reduce the background of cells having incorrectly integratedtargeting construct sequences, a combination positive-negative selectionscheme is typically used (Mansour et al. (1988) op.cit., incorporatedherein by reference). Generally, targeting constructs of the inventionpreferably include: (1) a positive selection expression cassette flankedby two homology regions that are substantially identical to host cellendogenous IMPA2 gene sequences, and (2) a distal negative selectionexpression cassette. However, targeting constructs which include only apositive selection expression cassette can also be used. Typically, atargeting construct will contain a positive selection expressioncassette which includes a neo gene linked downstream (i.e., towards thecarboxy-terminus of the encoded polypeptide in translational readingframe orientation) of a promoter such as the HSV tkpromoter or the pgkpromoter. More typically, the targeting transgene will also contain anegative selection expression cassette which includes an HSV tkgenelinked downstream of a HSV tkpromoter.

It is preferred that targeting constructs of the invention have homologyregions that are highly homologous to the predetermined targetendogenous DNA sequence(s), preferably isogenic (i.e., identicalsequence). Isogenic or nearly isogenic sequences may be obtained bygenomic cloning or high-fidelity PCR amplification of genomic DNA fromthe strain of nonhuman animals which are the source of the ES cells usedin the gene targeting procedure.

Vectors containing a targeting construct are typically grown in E. coliand then isolated using standard molecular biology methods, or may besynthesized as oligonucleotides. Direct targeted inactivation which doesnot require prokaryotic or eukaryotic vectors may also be done.Targeting transgenes can be transferred to host cells by any suitabletechnique, including microinjection, electroporation, lipofection,biolistics, calcium phosphate precipitation, and viral-based vectors,among others. Other methods used to transform mammalian cells includethe use of Polybrene, protoplast fusion, and others (see, generally,Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., 1989,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which isincorporated herein by reference).

It is thus a further object of the present invention to provide the useof the IMPA2 knock out animals as a model for neuroplasticity, inparticular to study the effects of enhanced neuronal plasticity. Inparticular to study the effects of an increased adaptive response to astressor in an animal model. Such transgenic animals can be commerciallymarketed to researchers, among other uses. The “knock out animal” or“transgenic animal” as used herein refers to a non-human animal, usuallya mammal and in particular a rodent, mice, having a 20- non-endogenous(i.e. heterologous) nucleic acid sequence present as an extrachromosomalelement in a portion of its cells or stably integrated into its germline DNA. This heterologous nucleic acid is introduced into the germline of said transgenic animal by genetic manipulation of, for example,embryos or embryonic stem cells of the host animal using art knownprocedures.

This and further aspects of the present invention will be discussed inmore detail hereinafter.

Experimental

Material and Methods

IMPA2 Knockout Mice

IMPA2 knockout mice were obtained from Lexicon Genetics Inc. and weregenerated from OST203987. The gene-trap was established with vectorVICTR48 and insertion occurred within intron 1 (FIG. 1).

Real-Time Quantitative Reverse Transcription (RTQ) PCR Analysis of IMPA1and IMPA2.

Total RNA isolated from different tissues dissected from wild type mouse(brain, liver, spinal cord, stomach, kidney, spleen, colon, lung, heart,oesophagus, pancreas, ileum) were analysed using real time quantitativePCR analysis for the tissue distribution of IMPA1 and IMPA2.

First strand cDNA synthesis was performed on 1 μg total RNA using randomhexamer primers and Superscript II RT (Invitrogen Life Technologies).Quantitative PCR was performed on an ABIPrism 7000 cycler (AppliedBiosystems) using a Taqman PCR kit. Serial dilutions of cDNA were usedto generate standard curves of treshold cycles versus the logarithms ofconcentrations for β-actin. The RTQ specific primer pairs and probes areenlisted herein below.

Mouse IMPA2 Selection of RTQ primers and probe: IMPA2_YW 5′-GAG GTG GCCGTG CAG TTG-3′ (SEQ ID No. 7) IMPA2_REV 5′-AGA CGC GTT TTT CCT CTGTCA-3′ (SEQ ID No. 8) IMPA2_Probe 5′-CCT GAT GAT TTG TCC CGC ACG CA-3′(SEQ ID No. 9) [5′ FAM] [3′ TAMRA]

Mouse IMPA1 Selection of RTQ primers and probe: IMPA1_FW 5′-AGC TGT TTCAAT TGG CTT CCT T- (SEQ ID No. 10) 3′ IMPA1_REV 5′-GCC GGT GTA CAT CTTATC TTC CA- (SEQ ID No. 11) 3′ IMPA1_Probe 5′-TGA ATA AAG AGA TGG AGTTTG GAA (SEQ ID No. 12) TTG TGT ACA GCT-3′ [5′ FAM] [3′ TAMRA]

Samples were run in triplicate and results are displayed only whencomplying with quality standards. Expression levels in the differentmouse tissues are expressed as relative levels after normalisation tomouse β-actin (FIG. 2) and as average CT-values (FIG. 3).

Phenotypical Analysis: Mouse Behavioural Tests

A panel of 10 wild type (+/+), 12 heterozygous (+/−) and 11 homozygous(−/−) IMPA2 mice, a mixture of males and females, were subjected tothree behavioural tests: Elevated Zero Maze (an art known method toevaluate anxiety-related behaviour), Porsolt Forced Swim test (an artknown method to evaluate depression-related behaviour) and Open FieldTest (an art known method to evaluate locomotor activity).

Elevated Zero Maze

The Elevated Zero Maze was performed during the dark phase of a normallight/dark cyclus. Each mouse was subjected to a 6-min testing sessionin the elevated zero maze. During this session, the following parameterswere recorded: total distance moved, relative distance in the open armsand relative duration in the open arms (FIG. 4). An unadjustedWilcoxon-Mann-Whitney rank sum test was used as statistical analysis ofthe data obtained (table 1). TABLE 1 Descriptive statistics and p-valuesobtained using an unadjusted Wilcoxon-Mann-Whitney rank sum test for thedata from the elevated zero maze in the IMPA2 mice Parameter Genotype NMean St Dev Median Total distance moved +/+ 10 1111 233 1078 +/− 12 1200245 1214 −/− 11 1237 185 1201 Parameter Group 1 Group 2 P-value Totaldistance moved +/+ +/− 0.381 +/+ −/− 0.1517 Parameter Genotype N Mean StDev Median Relative distance open arms +/+ 10 19.35 8.64 18.28 +/− 1228.25 9.36 29.46 −/− 11 26.55 9.4 24.09 Parameter Group 1 Group 2P-value Relative distance open arms +/+ +/− 0.0358 +/+ −/− 0.061Parameter Genotype N Mean St Dev Median Relative duration open arms +/+10 21.26 7.89 20.81 +/− 12 28.41 7.58 27.85 −/− 11 29.57 7.57 27.39Parameter Group 1 Group 2 P-value Relative duration open arms +/+ +/−0.0426 +/+ −/− 0.0357

Porsolt Forced Swim Test

The Porsolt Forced Swim test was carried out over 2 days. On the firstday, IMPA2 mice were subjected to a 10-minutes swimming session, ofwhich the first 6 minutes were recorded. 24 hours later, the same miceunderwent a second swimming session, this time for 6 minutes, of which 6minutes were recorded. Each recording period is divided in two separatetime intervals: 0→180 sec and 180→360 sec. The Porsolt Forced Swim testwas performed during the light phase of a normal light/dark cyclus.During the two sessions, the following parameters were recorded:immobility duration during the first 180 sec and immobility durationduring the last 180 sec (FIGS. 5 and 6). An unadjustedWilcoxon-Mann-Whitney rank sum test was used as statistical analysis ofthe data obtained (table 2 and 3). TABLE 2 Descriptive statistics andp-values obtained using an unadjusted Wilcoxon-Mann-Whitney rank sumtest for the data from the Forced Swim test in the IMPA2 mice on day 1Parameter Genotype N Mean St Dev Median relative immo duration +/+ 1055.13 20.08 55.86 0-180 sec +/− 12 48.56 20.41 51.06 −/− 11 45.96 15.3849.44 Parameter Group 1 Group 2 P-value relative immo duration +/+ +/−0.539 0-180 sec +/+ −/− 0.259 Parameter Genotype N Mean St Dev MedianRelative immo duration +/+ 10 62.33 24.64 71.36 180-360 sec +/− 12 69.4619.17 75.94 −/− 11 58.57 23.69 62.17 Parameter Group 1 Group 2 P-valueRelative immo duration +/+ +/− 0.445 180-360 sec +/+ −/− 0.523 ParameterGenotype N Mean St Dev Median Relative immo duration +/+ 10 58.73 21.0561.56 0-360 sec +/− 12 59.01 18.6 61.08 −/− 11 52.27 18.23 61.08Parameter Group 1 Group 2 P-value Relative immo duration +/+ +/− 0.9230-360 sec +/+ −/− 0.349

TABLE 3 Descriptive statistics and p-values obtained using an unadjustedWilcoxon-Mann-Whitney rank sum test for the data from the Forced Swimtest in the IMPA2 mice on day 2 Parameter Genotype N Mean St Dev Medianrelative immo duration +/+ 10 82.31 10.49 82.72 0-180 sec +/− 12 70.7514.41 70.94 −/− 11 71.28 12.33 70.67 Parameter Group 1 Group 2 P-valuerelative immo duration +/+ +/− 0.0375 0-180 sec +/+ −/− 0.0513 ParameterGenotype N Mean St Dev Median Relative immo duration +/+ 10 80.27 9.5977.97 180-360 sec +/− 12 73.8 17.1 79.03 −/− 11 73.75 13.31 77.97Parameter Group 1 Group 2 P-value Relative immo duration +/+ +/− 0.7223180-360 sec +/+ −/− 0.4679 Parameter Genotype N Mean St Dev MedianRelative immo duration +/+ 10 81.29 9.55 79.46 0-360 sec +/− 12 72.2814.56 73.78 −/− 11 72.52 12.36 76.14 Parameter Group 1 Group 2 P-valueRelative immo duration +/+ +/− 0.203 0-360 sec +/+ −/− 0.1734

Open Field Test

The Open Field Test was performed during the light phase of a normallight/dark cyclus. Each mouse was subjected to a 30-min testing sessionin an automated open field system. Locomotion in the horizontal andvertical pane was recorded. During this session, the followingparameters were recorded: time spent in center, distance travelled incenter, total distance travelled, number of moves, duration of moves,number of rearings and duration of rearings (FIG. 7). An unadjustedWilcoxon-Mann-Whitney rank sum test was used as statistical analysis ofthe data obtained (table 4). TABLE 4 Descriptive statistics and p-valuesobtained using an unadjusted Wilcoxon-Mann-Whitney rank sum test for thedata from the Open Field test in the IMPA2 mice Parameter Genotype NMean St Dev Median relative time spent +/+ 10 17.82 9.072 16.6 center+/− 12 14.8 4.5 14.4 −/− 11 13.13 4.95 14.36 Parameter Group 1 Group 2P-value relative time spent +/+ +/− 0.5387 center +/+ −/− 0.2816Parameter Genotype N Mean St Dev Median relative distance +/+ 10 20.137.33 19.69 travelled center +/− 12 18.29 3.85 16.83 −/− 11 17.13 4.8817.06 Parameter Group 1 Group 2 P-value relative distance +/+ +/− 0.5387travelled center +/+ −/− 0.3494 Parameter Genotype N Mean St Dev Mediantotal distance travelled +/+ 10 4099 960 3925 +/− 12 4207 1047 4113 −/−11 3801 1164 3820 Parameter Group 1 Group 2 P-value total distancetravelled +/+ +/− 0.7223 +/+ −/− 0.6047 Parameter Genotype N Mean St DevMedian total number of moves +/+ 10 605.2 40.58 613.5 +/− 12 580.4 55.06592 −/− 11 595.1 48.01 607 Parameter Group 1 Group 2 P-value totalnumber of moves +/+ +/− 0.2473 +/+ −/− 0.6415 Parameter Genotype N MeanSt Dev Median total duration of moves +/+ 10 1034 110.5 1006 +/− 12 1013107.6 1052 −/− 11 971 152.7 1009 Parameter Group 1 Group 2 P-value totalduration of moves +/+ +/− 0.9742 +/+ −/− 0.5116 Parameter Genotype NMean St Dev Median total number of rearings +/+ 10 105.3 42.3 88 +/− 1296.42 42.04 97 −/− 11 78.36 40.48 71 Parameter Group 1 Group 2 P-valuetotal number of rearings +/+ +/− 0.7105 +/+ −/− 0.0877 ParameterGenotype N Mean St Dev Median total duration of rearings +/+ 10 186.692.89 154 +/− 12 163.7 72.86 158.5 −/− 11 150.2 91.18 139.5 ParameterGroup 1 Group 2 P-value total duration of rearings +/+ +/− 0.7223 +/+−/− 0.223

Real-Time Quantitative Reverse Transcription (RTQ) PCR Analysis ofMIP-synthas and BDNF in Non-stressed Versus Mild Stressed Impa2 KO Miceand Wild Type Littermates.

Impa2 KO mice and WT littermates were subjected to restrained stress for7 consecutive days. On day 1, mice were stressed for 6 hours. On day 2to 7, mice were stressed for 1 hour daily. On day 8, mice weredecapitated, brains were removed and the hippocampus was carefullydissected out. WT and KO mice not subjected to stress were used ascontrols. Total RNA was isolated from hippocampus. RTQ PCR was performedas described in paragraph ‘Real time quantitative reverse transcription(RTQ) PCR analysis of IMPA1 and IMPA2.’

The RTQ specific primer pairs and probes are enlisted below. MIPsynthase_FW 5′-CTGCGCCTTCCTCAATGG-3′ (SEQ ID No. 13) MIP synthase REV5′-GCTGCGAAGCCAGTTCCA-3′ (SEQ ID No. 14) MIP synthase_Probe5′-TCCCCACAGAACACACTGGTACCCG (SEQ ID No. 15) [5′]6_FAM [3′]TAMRA BDNF_FW5′-CGGGACGGTCACAGTCCTA-3′ (SEQ ID No. 16) BDNF_REV5′-CACTTGGTCTCGTAGAAATACTGCTT-3′ (SEQ ID No. 17) BDNF_Probe5′-AGAAAGTCCCGGTATCCAAAGGCCAAC (SEQ ID No. 18) [5′]FAM[3′]TAMRA

A Two Way Analysis of Variance was used to analyze the data obtained,followed by Wilcoxon-Mann-Whitney rank sum post hoc analysis. In casedata were not normally distributed, data were normalized bylog-transformation.

Results

Real-Time Quantitative Reverse Transcription PCR Analysis of IMPA1 andIMPA2.

Real-Time Quantitative Reverse Transcription PCR of IMPA2 and IMPA1 inmultiple mouse tissues showed a wide tissue distribution, includingbrain (FIG. 2). Additionally, expression levels of mouse IMPA1 aregenerally higher than expression levels of mouse IMPA2 (FIG. 3).

Real-Time Quantitative Reverse Transcription (RTQ) PCR Analysis ofMIP-synthas and BDNF in Non-stressed Versus Mild Stressed Impa2 KO Miceand Wild Type Littermates.

It was found that MIP synthase expression levels are higher in IMPA2 KOmice compared to the WT littermates. Further, there is a significantup-regulation of MIP synthase in both the IMPA2 KO mice and the WTlittermates in response to stress (FIG. 8).

Also for BDNF the genotypic expression levels are higher in IMPA2 KOmice compared to WT littermates and is there a significant up-regulationin stressed versus non-stressed animals (FIG. 9).

Neither for MIP synthase, nor for BDNF, a significant synergy betweengenotypic expression levels and stress induced expression levels wasfound.

Phenotypical Analysis: Mouse Behavioural Tests

Elevated Zero Maze

A significant increase in the relative distance traveled in the openarms is observed in the IMPA2 +/− mice compared to their wild typelittermates (p=0.0358). We also see a trend in an increased relativedistance traveled in the open arms in the IMPA2 −/− mice compared totheir WT littermates (p=0.061). Under the anxiety-inducing conditions ofthe elevated zero-maze, IMPA2 −/− and +/− mice spent a significantlonger time in the “anxiogenic” open arms compared to their WTlittermates (resp. p=0.0357 and p=0.0426).

Porsolt Forced Swim Test

In the stress-inducing conditions of the forced swim test, nosignificant differences were observed between IMPA2 −/−, +/− and WTlittermates during the first 6 min of a 10-min swim session on the firstrecording day (table 2). However, this stressful experience on day 1rendered the IMPA2 −/− and +/− mice with an advantage on the secondrecording day. I.e. a significant decrease in immobility time wasobserved in the IMPA2 −/− and +/− mice compared to their WT littermatesduring the first 3 min of a 6-min swim session (resp. p=0.0513 andp=0.0375; table 3).

Open Field Test

No significant differences were found between IMPA2 −/−, +/− and WTlittermates in an open field test for all parameters studied.

Discussion

Myo-inositol monophosphatase 2 (IMPA2) is one of the key enzymes actingin the phosphatidyl inositol signalling pathway. Lithium, the simplestmood-stabilizing drug, inhibits both IMPA1 and IMPA2, key enzymes in thesynthesis and recycling of inositol. Additionally, a susceptibilitylocus for bipolar disorder is mapped on chromosome 18 p, in the regionwhere IMPA2 is located. To further evaluate a potential biological roleof IMPA2 in the field of affective spectrum disorders, an IMPA2 knockout was generated (Lexicon Genetics Inc.) and evaluated.

Real-Time quantitative reverse transcription PCR of IMPA2 in multiplemouse tissues showed a wide tissue distribution, including brain.However, expression levels are relatively low compared to IMPA1 levels.Anxiety- and depression-related behaviour of IMPA2-deficient mice weretested in an open field, an elevated zero-maze and in forced swimming. Agene-dosage effect was seen in the zero maze, where IMPA2 −/− mice spentmore time on the open areas of the maze than the WT littermates, but didnot differ in locomotor activity. No significant differences wereobserved between IMPA2 −/−, +/− and WT littermates during a first forcedswimming session, but a significant decrease in immobility was observedin the IMPA2 −/− and +/− mice compared to the WT littermates during asecond session 24 h later. No differences were found between IMPA2 −/−,+/− and WT littermates exploring an open field for 30 min.

In summary, the results presented here indicate that IMPA2 knockout miceshow reduced anxiety- and depression-related behaviour, but do notdiffer from WT littermates in locomotor function and point to a possiblerole for IMPA2 in affective disorders.

To confirm the initial hypothesis that IMPA2 may have a role inaffective disorders, i.e. IMPA2 KO mice suggested a mild antidepressantand anxiolytic phenotype, a further molecular characterization wascarried out by studying hippocampal expression changes of MIP synthaseand BNDF. MIP synthase is a gene involved in the inositol signalingpathway known to be play a role in manic depression and BDNF is a genein the neurotrophic signaling pathways, known to be involved inappetitive behaviour and in the development of a depression-likephenotype.

The stress induced up-regulation of MIP synthase and BDNF in IMPA2 KOmice and WT littermates together with the genotypic upregulation inIMPA2 KO, support a role for both the inositol and neurotrophic pathwayin the adaptive response to a stressor and suggest that the IMPA2 KO hasan improved adaptive potential (non-stressed condition) and response toa stressor.

References

Berridge, M. J. and Irvine, R. F., 1989. Inositol phosphates and cellsignalling. Nature 341, pp.197-205.

Berridge, M. J., Downes, C. P. and Hanley, M. R., 1989. Neural anddevelopmental actions of lithium: a unifying hypothesis. Cell 59, pp.411-419.

McAllister, G., Whiting, P., Hammond, E. A., Knowles, M., Atack, R.,Bailey, J. R., Maigetter, R. and Ragan, C. I., 1992. cDNA cloning ofhuman and rat brain myo-inositol monophosphatase. Expression andcharacterization of the human recombinant enzyme. Biochem. J. 284, pp.749-754.

Sjøholt, G., Gulbrandsen, A. K., Løvlie, R., Berle, J. Ø., Molven, A.and Steen, V. M., 2000. A human myo-inositol monophosphatase gene(IMPA2) localized in a putative susceptibility region for bipolardisorder on chromosome 18p11.2: genomic structure and polymorphismscreening in manic-depressive patients. Mol. Psychiatry 5, pp. 172-180.

Steen, V. M., Gulbrandsen, A. K., Eiken, H. G. and Berle, J.ø., 1996.Lack of genetic variations in the coding region of the myo-inositolmonophosphatase gene in lithium-treated patients with manic depressiveillness. Pharmacogenetics 6, pp. 113-116.

Yoon I S, Li P P, Siu K P, Kennedy J L, Cooke R G, Parikh S V, Warsh JJ., 2001. Altered IMPA2 gene expression and calcium homeostasis inbipolar disorder. Mol Psychiatry. 6, pp. 678-83.

Yoshikawa, T., Padigaru, M., Karkera, J. D., Sharma, M., Berrettini, W.H., Esterling, L. E. and Detera-Wadleigh, S., 2000. Genomic structureand novel variant of myo-inositol monophosphatase 2 (IMPA2). Mol.Psychiatry 5, pp. 165-171.

1. Use of an isolated IMPA2 protein in an assay to identify anti-anxietyor anti-depression compounds, wherein said compounds are characterizedin that they are capable of enhancing neuronal plasticity.
 2. Useaccording to claim 1 wherein the IMPA2 protein is being selected from;i. mouse IMPA2 (SEQ ID No:4), rat IMPA2 (SEQ ID No:6), human IMPA2 (SEQID No:2) or a functional fragment thereof, or ii. an amino acid sequenceencoding an IMPA2 protein, wherein said amino acid sequence has at least80% sequence identity, preferably at least 90% sequence identity, morepreferably at least 95% or most preferably at least 98% sequenceidentity with the human IMPA2 protein (SEQ ID No:2) over its entirelength.
 3. Use of an isolated polynucleotide encoding an IMPA2 proteinin an assay to identify anti-anxiety or anti-depression compounds,wherein said compounds are characterized in that they are capable ofenhancing neuronal plasticity.
 4. Use according to claim 3 wherein theisolated polynucleotide encodes an IMPA2 protein in an assay accordingto the invention, wherein said IMPA2 protein is preferably beingselected from; i. polynucleotides encoding the mouse (EMBL:BC011093—SEQID No:3), rat (EMBL:AY160191—SEQ ID No:5) or human (EMBL:BC011093—SEQ IDNo:1) IMPA2 enzyme; or ii. a polynucleotide sequence encodig an IMPA2protein, wherein said amino acid sequence has at least 80% sequenceidentity, preferably at least 90% sequence identity, more preferably atleast 95% or most preferably at least 98% sequence identity with thepolynucleotide encoding for the human IMPA2 protein (SEQ ID No:1) overits entire length.
 5. A method to identify anti-anxiety oranti-depression compounds, wherein said anti-anxiety or anti-depressioncompounds are capable of enhancing neuronal plasticity, said methodcomprising the steps of: a) providing a composition comprising an IMPA2protein; b) contacting the IMPA2 protein with the test compound; and c)measuring the activity of the IMPA2 protein wherein a decrease in theIMPA2 activity in the presence of the test compound is an indicator ofan anti-anxiety or anti-depression compound.
 6. A method for determiningwhether a compound is capable of enhancing neuronal plasticity, saidmethod comprising the steps of: a) providing a composition comprising anIMPA2 protein; b) contacting the IMPA2 protein with the test compound;and c) measuring the activity of the IMPA2 protein wherein a decrease inthe IMPA2 activity in the presence of the test compound is an indicatorof a neuronal plasticity enhancing compound.
 7. A method according toclaim 5 wherein the activity of the IMPA2 protein is assessed bymeasuring the hydrolysis of myo-inositol 1-phosphate to generateinositol and inorganic phosphate
 8. A method according to claim 5wherein the activity of the IMPA2 protein is assessed by measuring theaccumulation of either myo-inositol monophosphate product in the form ofradiolabeled inositol or inorganic phosphate (Pi) in the form ofradiolabeled ³²Pi or in a colorimetric assay.
 9. A method according toclaim 5 wherein the compositions comprising the IMPA2 protein couldeither be cellular extracts, whole cells or organisms expressing theIMPA2 proteins according to the invention.
 10. A method of treating acondition associated with an impaired neuronal adaptive response,comprising the step of administering an effective amount of an IMPA2inhibitor to a subject in need of such treatment.
 11. A method accordingto claim 9 wherein the conditoion associated with an impaired neuronaladaptive response is selected from the group consisting of memorydysfunction or neurodegenerative diseases.
 12. A method according toclaim 11 wherein the neurodegenerative diseases are selected from thegroup consisting of senile dementia, Alzheimer's disease, Parkinsosn'sdisease, Huntington's chorea, cerebellar-spinal adrenoleucodystrophy,pick's disease or Wilson's disease.
 13. A method of treating neuronaldamage, comprising the step of administering an IMPA2 inhibitor to asubject in need of such treatment.
 14. A method according to claim 13,wherein the neuronal damage is selected from the group consisting ofstroke, multi-infarct dementia, head trauma, cerebral ischemia, braininjury and neurodegenerative diseases.
 15. A method of enhancing memoryand learning, comprising the step of administering an effective amountof an IMPA2 inhibitor to a subject in need of such treatment.
 16. Theuse of a compound identified in an assay according to claim 1, in thepreparation of a medicament for treating anxiety or in the preparationof a medicament for promoting neuronal plasticity.
 17. The use of anIMPA2 knock out animal as a model for neuroplasticity.